Liver cancer is one of the leading causes of cancer death world wide. One of the critical signaling pathways that are activated in human HCCs is the Ras/MAPK pathway. However, in human HCCs, mutations of Ras genes are very rare. Using genomic analysis, we identified that Spry2 and EphA2, both inhibitors of Ras/MAPK pathway, are down-regulated and may function as tumor suppressors of HCC. In this application, we hypothesize that loss of Spry2 and/or EphA2 expression leads to activation of Ras/MAPK signaling and cooperates with activated beta-catenin to induce HCC in vivo. To test this hypothesis, we will develop and utilize a novel murine model for liver cancer by integrating shRNA mediated gene silencing and hydrodynamic transfection to specifically knockdown target gene expression in mouse hepatocytes. In this application, we propose the following two aims: Aim 1: To determine whether loss of Spry2 cooperates with N90-2-catenin to promote liver cancer development in mice;and Aim 2: To determine whether loss of EphA2 cooperates with N90-2-catenin to promote liver cancer development in mice. In these two aims, we will generate specific hydrodynamic transfection constructs that express shRNA against mouse Spry2 or EphA2 and co-express them together with N90-2-catenin in mice and monitor for liver tumor development. In addition, we will use conditional Spry2 or EphA2 knockout mice as the comparison for shRNA gene silencing in promoting liver tumor development. Together, in this exploratory R21 application, we will illustrate the roles of Spry2 and EphA2 as tumor suppressors in HCC and identify possible novel mechanisms for activation of the Ras/MAPK pathway in the absence of Ras mutations. In addition, we will expand our in vivo gene transfection technology in generation of novel sporadic and flexible murine models for hepatic carcinogenesis. These novel in vivo transfection technologies can be integrated into oncogenomics studies and will enable us to efficiently analyze the functional significance and genetic interactions of genes identified from genomic analysis in vivo. The proposed study will therefore provide novel insight into the molecular genetics underlying human HCC pathogenesis Public Health Relevance: Liver cancer is a deadly disease, lacking any effective treatment options. Liver cancer incidence is increasing in the US. In this application we will develop new mouse models for liver cancer and study the role of two genes in liver tumor development. These studies will provide us tools and novel drug targets for the treatment of this malignancy.